In recent years, a highly competitive market place and environmental concerns have , encouraged researchers to develop technologies to improve the efficiency of livestock and dairy productions. However, a greater public awareness of animal welfare issues and of food production indicates that further improvement in efficiency may have to be achieved without compromising animal health and well-being. Recent findings in a number of animal systems suggest that epidermal growth factor (EGF) may be used in livestock and dairy productions as a feed additive to stimulate precocious maturation of gut cells to secrete an appropriate spectrum of digestive enzymes; increase nutrient absorption; and prevent or treat intestinal infection.
Derived from a precursor protein of about 1,200 amino acids, EGF is a 6 kDa polypeptide composed of 53 amino acids (Carpenter and Cohen, 1979). It is naturally present in saliva, intestinal secretions, and other bodily fluids, and is produced in large quantities in colostrum and milk (Donovan and Odle, 1994). EGF stimulates the growth and maturation of the human fetal stomach. Due to high EGF concentrations in the colostrum of most species, and the presence of EGF receptors within the intestine, EGF (and other growth factors) have been proposed to also contribute to early postnatal gastrointestinal development (Donovan and Odle, 1994).
EGF may be involved in regulating nutrient uptake. In the rodent, EGF increases electrolytes, glucose and proline transport across jejunal brush-border membranes. In the piglet, EGF promotes maturation of gut cell function by enhancing both sucrase and maltase activity without any marked effect on lactase and alkaline phosphatase activities (International Publication No. WO 88/04180 to Wilson et al.). Use of EGF as a feed additive for growth promotion is thus advantageous.
Enteric colibacillosis or scour is a bacterial infection caused by the pathogen Escherichia coli. Common in newborn and young farm animals, scour has considerable impact upon the agricultural economy. Overcrowding of young animals in confined areas is commonly followed by outbreaks of scour, which is characterized by diarrhea, dehydration, and eventual death. Dairy calves receiving milk replacer are more susceptible to scour than those fed cow's milk, with a morbidity rate due to infection of up to 75%.
Since a vaccine for scour is presently lacking, an effective treatment such as EGF is desirable. Supplementation with EGF improves intestinal functions of piglets infected with rotavirus (Zijlstra et al., 1994). Further, oral EGF administration reduces the rate of enteric infections in rabbits and prevents the reduction in weight gain caused by infection (Buret et al., 1997). U.S. Pat. No. 5,753,622 to Buret et al. discloses methods for treating scour and other pathogenic infections, and for increasing weight gain by administration of EGF orally or indirectly in animal feed. In most of these previous studies, EGF from a species different from the recipient was used; however, in principle, EGF of the same species would be preferable to avoid undesirable side effects. For use in dairy or beef cattle production, it is thus preferable to use bovine EGF (bEGF).
A feed additive or supplement must be economical to be widely adopted by producers. Due to advances in molecular biology and recombinant DNA technology, large quantities of foreign proteins for research, therapeutic, and industrial applications are efficiently produced. Genes coding for desired proteins can be transferred from organisms impractical for production into microbial, plant, or animal expression systems, with microbes most commonly used for expression of heterologous proteins due to their ease of growth and genetic manipulation. Human EGF has been expressed in yeast at a rate of 40 ng/mg of protein (U.S. Pat. No. 5,096,825 to Barr et al.), while mouse EGF has been produced by the yeast Pichia pastoris at a rate of 450 μg/ml of medium (Clare et al., 1991).
Complementary DNA sequences encoding the mature EGF protein have been previously cloned from mouse, rat, pig, horse and human (Gray et al., 1983; Simpson et al., 1985; Kim et al., 2001; Stewart et al., 1994; Bell et al., 1986). The deduced amino acid sequences show 55% to 85% identity to each other, with striking conservation of key structural residues, particularly three glycines (residues 18, 36, and 39), six cysteines (residues 6, 14, 20, 31, 33, and 42), and tyrosine 37. The variation in other residues presumably accounts for the very low cross-reactivity between species observed with antisera and nucleic acid probes. The cDNA sequence encoding the precursor protein has been cloned from human, pig, and mouse.
Although cDNA sequences encoding the mature EGF protein have been previously cloned from various sources as previously described, the DNA sequences encoding the mature EGF protein have not previously been obtained from a bovine source. However, bEGF may benefit dairy and beef cattle production by promoting growth; preventing or treating intestinal infections; increasing nutrient absorption; and accelerating development of immature gut cells. For such potential commercial applications, use of EGF from a bovine source itself is desired to avoid undesirable side effects which may compromise animal health and well-being. There is thus a need to obtain the DNA sequences encoding the mature bEGF protein and to produce bEGF successfully in a recombinant system.
In general, the homology between human and bovine sequences allows the use of human probes or primers to obtain the corresponding bEGF DNA sequence. However, sequences for the bEGF DNA and protein sequences were found to be very different from those of other species, which accounts for the initial difficulties in attempting to clone the bEGF DNA sequence, and explains why this sequence had not yet been successfully cloned despite the many promising commercial applications of such an invention.